The GLI genes, GLI1 and GLI2, encode transcription factors that regulate target genes at the distal end of the canonical Hedgehog signaling (HH) pathway (SHH->PTCH->SMO->GLI), tightly regulated in embryonic development, tissue patterning and differentiation, with low-level expression in adult tissues. In cancers, both GLI1 and GLI2 are oncogenes, aberrantly and constitutively activated. Oncogene-driven signaling pathways, in particular KRAS/BRAF in colon cancer, circumvent the HH-GLI axis, channel through GLI, and induce further constitutive GLI activation, giving GLI a pivotal role in cell survival. Using GANT61, a small molecule inhibitor of GLI-dependent transcription, we have demonstrated that targeting GLI terminates oncogenic HH-GLI and KRAS/BRAF-GLI signaling, inducing extensive cell death in seven human colon carcinoma cell line models examined. We have demonstrated that: 1) GANT61 is specific for targeting GLI; 2) inhibition of GLI1 and GLI2 by GANT61 rapidly reduces binding to consensus sequences in target gene promoters and inhibits GLI-dependent transcription; 3) GANT61 induces GLI-dependent transcriptional stalling of RNA Polymerase II (Pol II) at sites of early elongation with accumulation of RNA:DNA hybrids (R-loops); 4) consistent with transcriptional inhibition, GANT61 induces DNA damage (?H2AX foci) in S-phase and non-S-phase cells, recognized at the initiation of S-phase (G1/S), prior to the onset of cell death. The overall goal s to understand the mechanisms by which GANT61 inhibits GLI-dependent transcription that induces DNA damage leading to extensive cell death. In Specific Aim 1, we have identified stalling of Pol II adjacent to GLI binding regions at target gene promoters, associated with modification of promoter-bound pause (DSIF, NELF) or pause-release (P-TEFb) factors. Single-stranded DNA (R-loops) and RNA: DNA hybrids are detected at sites of stalled Pol II; ?H2AX foci are induced. The hypothesis is that GANT61-induced GLI-dependent stalling of Pol II creates ssDNA at sites of early elongation that generates DNA DSBs within R-loop regions. In Specific Aim 2, we have demonstrated that DNA damage is recognized at the initiation of S-phase, inducing a transient intra-S-phase checkpoint prior to the onset of cell death. The GLI1 target gene FOXM1, and its transcriptional target CDC6, that regulate the G1/S transition and initiation of DNA replication, are decreased in GANT61-treated cells. The hypothesis is that FOXM1 and CDC6 regulate Pre-Replication complex assembly at G1/S thereby inhibiting the initiation of DNA replication, which controls outcome of the GANT61 response, dependent on GLI. In Specific Aim 3, we will target constitutive GLI activation in uniquely engineered in vivo models of human colon cancer xenografts and in a transgenic mouse GLI-luciferase reporter model. GLI is constitutively activated in a wide variety of human cancers. Further, KRAS is mutated in 30% of all human cancers, and in 50% of colon carcinomas. This proposal therefore has the potential for high impact, and is anticipated to lead to new approaches and therapeutic strategies for GLI as a target.